TIM Crystals Grown by Capillary Counterdiffusion - American

CRYSTAL GROWTH & DESIGN

TIM Crystals Grown by Capillary Counterdiffusion: Statistical Evidence of Quality Improvement in Microgravity†

2007 VOL. 7, NO. 11 2161–2166

Christine Evrard,*,‡,|,# Dominique Maes,§,# Ingrid Zegers,§,⊥ Jean-Paul Declercq,| Celine Vanhee,§ Joseph Martial,‡ Lode Wyns,§ and Cécile Van De Weerdt‡ Laboratoire de Biologie Moléculaire et de Génie Génétique, UniVersité de Liège, Batiment B34, AVenue de l’Hopital 1, B-4000 Liège, Belgium, Ultrastructure Unit, Vrije UniVersiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, and Unité de Chimie Structurale, UniVersité Catholique de LouVain, Place Louis Pasteur 1, B-1348 LouVain-la-NeuVe, Belgium ReceiVed July 24, 2007; ReVised Manuscript ReceiVed September 19, 2007

ABSTRACT: The capillary counterdiffusion method is a very efficient crystallization technique for obtaining high-quality protein crystals. This technique requires a convection-free environment, which can be achieved using either gelled solutions, very thin capillaries, or microgravity conditions. To study the influence of a convection-free environment on protein crystal quality and to evaluate two different experimental implementations to achieve it, we have made a comparative analysis of crystals grown by capillary counterdiffusion in agarose, a convective-free environment on Earth, and crystals grown in microgravity at the International Space Station. Thermotoga maritima triose phosphate isomerase (TIM) was chosen as a model for this study. The statistical analysis reveals a significant improvement for the crystals grown in microgravity in terms of their Rmerge, B-value, and mosaicity, but the statistical evidence is insufficient to show a similar benefit for the resolution and mean intensity parameters. These results are quite surprising because it is known that, unlike gels, the noisy microgravity scenario offered by the ISS cannot sustain a convection-free environment on the time scale of days required for protein crystallization experiments. Introduction Growing crystals of quality still represents the major bottleneck in structural biology.3,4 The capillary counterdiffusion method developed by Garcia-Ruiz and collaborators in 1993 is a very efficient crystallization technique for obtaining highquality crystals.5,6 This technique is based on the counterdiffusion of protein and precipitant along a capillary. The diffusion process produces a wave of supersaturation with decreasing amplitude and increasing width that moves along the capillary. The system evolves through a continuous range of different supersaturation conditions, enabling simultaneous screening for optimal conditions for protein crystal growth. Uniform propagation of the supersaturation wave requires a convection-free environment, which can be achieved using either gels, very thin capillaries (micro fluidic systems), or microgravity. Silica and agarose gels have both been used to crystallize protein by counterdiffusion. At a concentration equal to 0.1%, agarose does not form a true gel but increases the viscosity to a value high enough to avoid convective flow and sedimentation.12 Comparison of crystals grown in gels with those grown in standard trials shows a significant improvement in crystalline order in spite of the incorporation of a large amount of gel into the crystal.8 The counterdiffusion technique was adapted for the first time for microgravity experiments in the Advanced Protein Crystallization Facility (APCF) and very high quality crystals of tetragonal hen egg-white lysozyme were grown in space during † Part of the special issue (Vol 7, issue 11) on the 11th International Conference on the Crystallization of Biological Macromolecules, Quebec, Canada, August 16–21, 2006 (preconference August 13–16, 2006). * Corresponding author. Phone: 32 (0)4 366 33 79. Fax: 32 (0)4 366 41 98. E-mail: [email protected]. ‡ Université de Liège. § Vrije Universiteit Brussel. | Université Catholique de Louvain. ⊥ Present work address: Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, B-2440 Geel, Belgium. # These authors contributed equally to the work in this paper.

the STS-95 mission of the NASA Space Shuttle.9 For a straightforward implementation of counterdiffusion experiments, researchers developed the Granada Crystallization Box (GCB).11 The GCB has now been validated as a passive, inexpensive, and high-density crystallization apparatus for growing protein crystals in microgravity. To fully exploit the strengths of this technique, it is necessary to complete our knowledge about the relationship between the quality of the crystals obtained and the environment in which they are produced: in gels on ground or in space. To this end, we performed experiments aboard the International Space Station (ISS) using GCB-type apparatuses. The model protein used for this study was Triose Phosphate Isomerase (TIM) from the thermophilic organism Thermotoga maritima. This protein is easily purified and stable over time, and its crystallization is highly reproducible.16 In this paper, we first describe the optimization steps to adapt the TIM crystallization conditions known from vapor diffusion experiments to the capillary counterdiffusion technique. We then analyze the distribution of crystal sizes, and finally, we compare X-ray quality parameters for crystals grown in two nonconvective environments, in solution in space, and in agarose-gelled medium on ground. These experiments were directed by the European Space Agency (ESA) and conducted within the framework of the Belgian (Odissea) and Spanish (Cervantes) taxi flight space missions in November 2002 and October 2003, respectively. Results Optimization of TIM Crystallization Conditions for Counterdiffusion. TIM was originally crystallized using the hanging drop vapor diffusion technique and its structure was solved to a resolution of 2.8 Å.16 To determine the optimal TIM crystallization conditions for our experiments, we first had to adapt these conditions to the counterdiffusion setup. Furthermore, as the GCB is a passive device that does not allow any

10.1021/cg700687t CCC: $37.00  2007 American Chemical Society Published on Web 10/26/2007

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Table 1. Delays for the Appearance of Crystals at Different Positions in the Capillary (diameter ) 0.5 mm); Positions in the Capillary Refer to the Distance along the Length of the Capillary as Measured from the end of the Capillary Inserted into the Base of the GCB delay for the appearance of the first crystal (days) position in the capillary (mm) 0–10 10–20 20–30 30–40

0 M AS protein (12 mg/mL)

0.5 M AS protein (12 mg/mL)

8 13 >14 >14

4 6 9 12

manual intervention, the parameters of the GCB experiments (protein/precipitant concentrations, capillary lengths and heights, etc.) had to be chosen in such a way that the precipitant and protein solutions would come into contact with each other only after a time period long enough for the samples to reach the ISS. Moreover, the crystallization process had to be completed in the 11 (Odissea) or 10 (Cervantes) days of the mission duration. In the original hanging drop crystallization, a protein solution of 8.5 mg/mL in 70 mM TRIS-HCl (pH 8.0), 2 mM EDTA, and 400 mM NaCl was mixed with an equal amount of reservoir solution consisting of 100 mM TRIS-HCl buffer with 2.0 M ammonium sulfate (AS). For the first counterdiffusion experiment, we used a protein concentration of 5 mg/mL with the protein solution in 0.1% agarose gel. The experiment was set up such that at the end of the precipitant diffusion process, the ammonium sulfate concentration in the capillary was 2 M. Seventeen days were needed to observe the first crystals inside the capillaries, a period too long for the space missions. The capillary counterdiffusion technique has a longer induction time than the vapor diffusion method, which can be explained by the differences in the manner in which precipitant and protein come into contact and the rate at which the supersaturation is achieved.26 To shorten the nucleation induction time, we increased the protein concentration to 12 mg/mL and included a small quantity of precipitant (final concentration, 0.5 M AS) with the protein solution, but still kept the whole solution below the threshold of TIM solubility. The delays of the appearance of crystals at different positions in the capillary were measured (Table 1). The results presented in Table 1 show that by increasing the protein concentration from 5 to 12 mg/mL in the capillary, the delay for the appearance of the first crystal decreases from 17 to 8 days. Moreover, when a small quantity of precipitant is added in the protein solution, the delay is reduced to 4 days. Comparison of Crystals Grown in Two Nonconvective Environments: Microgravity and Gelled Solution on Ground. (a) Analysis of the Distribution of Crystal Sizes along the Capillary. Before studying the relationship between crystal quality and the type of convection-free environment, we carefully measured the crystal size at different positions in the capillaries for the two growth environments (gel on ground and ISS-microgravity without gel) (Figure 1). A total of 171 TIM crystals were used. The size of the crystals was determined under a microscope. In general, the three dimensions of the crystals are similar. Therefore, the two visual dimensions were measured and their average was taken as the size variable. The analysis reveals an increase in crystal size along the length of the capillaries. Indeed, in the counterdiffusion technique, a wave of supersaturation moves along the capillary with decreasing amplitude and rate. Low supersaturation and

Figure 1. Size distribution of ground (green) and space (blue) grown crystals along the capillary. The position in the capillary is measured from the end inserted into the base of the GCB. The sizes are given as boxplots (50% of the data are within the box, the whiskers indicate the maximum and minimum value (outliers omitted)). The outliers are indicated with their crystal number as circles (values between 1.5 and 3 box lengths from the upper or lower edge of the box. The box length is the interquartile range) and asterisks (values more than 3 box lengths from the upper or lower edge of the box).

high protein concentration are the most optimal conditions for crystal growth. For space experiments, this counterdiffusion profile may become disturbed by the movement of the crystals because of the reentry in the Earth atmosphere and the g-jitters and residual accelerations in the ISS.2,7,17,19,22,25 However, in our experiment, most of the crystals grew attached to the walls of the capillaries and kept their positions there even after reentry to the Earth’s atmosphere. On the whole, the size of the space crystals is larger than the size of the ground crystals. (Mann–Whitney: p-value ) 0.005 for the 171 crystals) (see Figure 2 a). (b) Analysis of Crystal Quality Parameters. From among the 171 crystals, 34 were chosen on the basis of their size as being suitable for X-ray diffraction: 16 from the space experiments and 18 from ground. To have the same diffracting volume, we chose same size crystals from the two groups (Mann–Whitney test: p-value 0.60, see Figure 2 b). For the X-ray data quality comparison, data sets were collected in the same experimental setup on cryocooled crystals. One of the advantages of the capillary counterdiffusion method is that crystals can be X-rayed in situ, eliminating the need for crystal handling. In our case, however, a large number of crystals had to be measured in order to get relevant statistical results. Because of the important density of crystals obtained in the capillaries, it was impossible to measure enough isolated crystals directly from the capillaries. Moreover, as we wanted to compare the crystal quality parameters of complete X-ray data sets, we used a cryoprotectant to minimize the radiation damages. The 34 TIM crystals measured all belonged to space group P3212, having a packing very similar to the one obtained by the hanging drop method.16 The latter belong to space group P3221, with a much larger unit-cell size. A change of space group in counterdiffusion setups has been observed for several proteins. It can be attributed to the different rates at which supersaturation is achieved and is the subject of another paper.26 Data sets for the 34 crystals were analyzed for signal-to-noise ratio mean (I/σ(I)), mosaicity, B-value, resolution, and Rmerge. The SCALEPACK output20 for

Crystal Quality Improvement in Microgravity

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Figure 2. Boxplots of the sizes of ground (green) and space (blue) grown crystals for a) all the crystals and b) the crystals used for the statistical analysis. The p-values of the Mann–Whitney comparison test are indicated below each figure.

all the data to 2.2 Å was used for this analysis. As most of the crystals diffracted to a higher resolution, the parameter indicating the resolution was chosen as the value corresponding to a mean (I/σ(I)) equal to 10 (see Figure 4). The obtained values are reported in Table 2 and schematically represented as boxplots in Figure 3. Note that in Table 2, the values of the two missions are reported separately. For the statistical analysis, both missions

are taken together in order to have a larger sampling. On the one hand, a larger sample size makes it easier to see statistically significant differences, but it also leads to a greater variability in the diffraction parameters. It is clear that the five parameters under investigation are correlated. The correlation matrix is given in Table 3, with the normal Pearson correlation as well as the more robust Spearman correlation indicated. There is a significant correlation at the 5% level between mosaicity, signal-to-noise ratio, and resolution. Note that both the average values (Table 2) and the medians (Figure 3) for all the crystal quality parameters are better for the space-grown than the ground-grown crystals. To check if the improvement was statistically significant, we performed a two-sided Mann–Whitney test, a robust statistical test for comparing a parameter between two different populations. This comparative statistical analysis showed improved mosaicity (pvalue ) 0.03), B-value (p-value ) 0.02), and Rmerge (p-value ) 0.01) for the space crystals at a 5% significance level. The available data showed no difference for the signal-to-noise ratio (mean (I/σ(I))) (p-value ) 0.10) or the resolution (p-value ) 0.17) at a 5% significance level. This does not imply that there is no difference but that any difference present is too small to be detected with this limited data set. The only parameter for which the null hypothesis of equality at a 10% significance level is not rejected is the resolution. Note that if we would have opted for a one-sided test with an alternative hypothesis of better values (lower mosaicity, lower B-value, lower Rmerge, lower signal-to-noise ratio, and higher resolution) for the space-grown crystals, the calculated p-values would have been halved. From the boxplots (Figure 3), it is clear that the interquartile range, a robust measure for variation in data, is larger in the ground crystals than the space crystals for the signal-to-noise ratio, Rmerge, and resolution parameters. Figure 4 gives a detailed picture of the signal-to-noise ratio as a function of resolution for all the crystals from both the ground controls and space missions. It is clear that most space crystals are superior to ground crystals over the whole resolution range measured, despite the higher p-value (p ) 0.10) for the two-sided Mann–Whitney test. Note that one space crystal of the Odissea mission is of inferior quality. This crystal was flagged as an outlier in Figure 3 (crystal 11). All the crystals analyzed in this study diffracted X-rays to a resolution better than or equal to 2.2 Å. This is a large improvement compared to the 2.85 Å that corresponds to the resolution of the best diffracting crystal obtained by hanging drop experiments.16 With a longer exposure time on the same beamline, one of the best crystals diffracted to 1.7 Å. The improvement in the diffraction power can be attributed to a better crystal packing and at least partly to the change in space group. Our results suggest that counterdiffusion is a powerful

Table 2. Comparison of the Data Quality Parameters for Agarose and Microgravity-Grown Crystals (standard deviations are given in parentheses) Odissea

mean (I/σ(I)) Wilson B (Å2) Rmerge mosaicity (deg) resolution (Å) at mean (I/σ(I)) )10

Cervantes

microgravity (5 crystals)

ground controls; agarose gel (0.1%) (9 crystals)

microgravity (11 crystals)

ground controls; agarose gel (0.1%) (9 crystals)

16.9 (4.8) 34 (3) 0.07 (0.03) 0.30 (0.05) 2.6 (0.4)

13.7 (2.9) 36 (3) 0.08 (0.02) 0.38 (0.05) 2.7 (0.2)

12.8 (2.6) 35 (3) 0.07 (0.01) 0.40 (0.10) 2.8 (0.3)

8.9 (1.7) 35 (2) 0.09 (0.01) 0.49 (0.13) 3.3 (0.6)

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Figure 3. Comparative boxplots between ground (green) and space (blue) crystals for the X-ray quality parameters (a) B-value, (b) mosaicity (deg), (c) mean (I/σ(I), (d) Rmerge, and (e) resolution. The p-values of the Mann–Whitney comparison tests are indicated below each figure. Table 3. Correlations between the X-Ray Quality Parameters; Pearson Correlation Is Given above the Diagonal and the More Robust Spearman Correlation below the Diagonal

B-value mosaicity mean (I/σ(I)) Rmerge resolution

B-value

mosaicity

mean (I/σ(I))

Rmerge

resolution

1.000 0.527 -0.541 0.573 0.495

0.640 1.000 -0.871 0.651 0.883

-0.558 -0.786 1.000 -0.837 -0.965

0.539 0.560 -0.811 1.000 0.756

0.589 0.938 -0.857 0.700 1.000

technique to obtain new crystal forms with better X-ray characteristics.26 Discussion Protein crystallization experiments in microgravity have now been conducted for more than two decades, and a large number of reviews have been dedicated to compile the results and analyze the influence of experimental variables on crystal quality.13,15,18,23,24 It is worth noting that most comparative studies have been conducted between a microgravity environment in space and a convective environment on ground. Furthermore, in most studies, one is reporting on the improvement of a specific quality parameter (resolution, Rmerge, etc.) for a very limited number of crystals (often only one). Our study is the first to compare, with a robust statistical analysis of the X-ray crystal quality parameters, crystallization in the ISS microgravity environment with crystallization in a convectivefree environment on Earth. The statistical tests point to an improvement in X-ray quality parameters such as B-value,

Figure 4. Mean signal-to-noise ratio as a function of the resolution for the ground (green) and space (blue) grown crystals of (a) the Odissea mission and (b) the Cervantes mission.

Crystal Quality Improvement in Microgravity

mosaicity, and Rmerge for the space crystals. In our comparative study, we randomized the uncontrollable parameters such as crystal handling, cryocooling, etc. As a consequence, this leads to additional variations in the X-ray quality parameters, but there is no reason to believe that the effect of these parameters would be any different for the ground crystals than the space-grown ones. Nevertheless, this increase in variation of the data will unfortunately make it more difficult to spot statistically relevant differences between the two groups. The intention of our study was not to show that crystallization in ISS microgravity is better than crystallization in gels on earth. We wanted to compare crystals grown in gel, a convective-free environment on Earth, with crystals grown in the microgravity of the ISS, which is supposed to be a convection free environment and, in addition, gel-free. However, it is known that the microgravity offered by the ISS cannot sustain a convective free environment, because of g-jitters and residual acceleration. Large crystal movements, resulting in convective flows, have been observed in previous space experiments and by our team in the ISS.25 It was therefore surprising that the crystallization in ISS microgravity gives similar or better results than the crystallization in gels on earth. For TIM, it turns out that several X-ray quality parameters are improved. In a convection-free environment, a protein depletion zone is induced around the growing crystal, which is believed to be beneficial for crystal quality.10,21 The depletion zone model suggests that the ground crystals grown in gels should be higher than or at least similar in quality to the space ones. Our results are quite intriguing because our space crystals are of higher quality than the ground crystals. The reason for this is not clear. If TIM has a very shallow depletion zone, we would expect crystals of equal quality. A possible explanation could be that incorporation of gel into the growing crystal is deleterious for the X-ray diffraction quality. The change in diffusion coefficient in gel versus a gel-free environment might also play a role. It is important to mention that the conclusions reported here apply only to TIM from Thermotoga maritima and that there is as yet no evidence that they are applicable to other proteins. To complement our results, experiments will be conducted in a new space mission planned by ESA for the end of 2007. A more elaborate statistical study in a better microgravity environment on a wide variety of proteins will be performed. Materials and Methods Protein Production and Purification. TIM from Thermotoga maritima was produced and purified as previously described1 with minor modifications. The recombinant gene of TIM was introduced in E.coli BL21 (DE3) cells, and the corresponding soluble protein was expressed after an overnight culture at 37 °C. The cells were harvested by centrifugation and resuspended in 100 mM TRIS-HCl buffer pH 8.0 and 2 mM EDTA. The cell suspension was passed twice through a French press and the cell debris was removed by centrifugation. To purify TIM, we took advantage of its thermostability. The supernatant was heated to 80 °C for 45 min and the denaturated proteins were removed by one further centrifugation step. The supernatant was filtered through a 0.22 µm cellulose filter (Millipore) and loaded on a 5 mL HiTrap Q HP sepharose column (GE Healthsciences) equilibrated with 100 mM Tris buffer pH 8.0, 2 mM EDTA. TIM was eluted with 10 column volumes 100 mM TRIS-HCl buffer pH 8.0, 2 mM EDTA, 0 to 1.0 M NaCl at a flow rate of 4 mL/min. The active fractions were pooled in a molecular porous membrane tube (Spectrum Laboratories) (cutoff, 6–8000 Da) and dialysed three times against 100 volumes of 100 mM TRIS-HCl buffer pH 8.0, 2 mM EDTA, 200 mM NaCl. NaCl is added to avoid reversible aggregation of TIM during the concentration step. For crystallization, the protein was concentrated with a Vivaspin ultrafiltration membrane (Vivascience).

Crystal Growth & Design, Vol. 7, No. 11, 2007 2165 Table 4. Summary of the Counterdiffusion Experiments

experiment protein concentration (mg/mL) AS concentration (M) is agarose included with the protein? precipitant solution outside the capillary (M AS) height of agarose gel layer in the GCB (mm) height of the precipitant layer poured onto the gel (mm) insertion depth of the capillary into the gel (mm) length of the capillaries (mm) diameter of the capillaries (mm)

optimization experiments& and Odissea Cervantes ground controls microgravity microgravity 12 0 to 0.5

12 0.5

24 0.25

yes 4

no 4

no 4

22

17

17

30

34

34

10

7

7

42

42

42

0.5

0.5

0.5

Capillary Counterdiffusion Experiments. The capillary counterdiffusion experiments were carried out in GCBs for the ground experiments or GCB-type apparatuses adapted for the space missions. For both the ground and space experiments, agarose gel was poured into the base of the GCB (as described below), but only the ground optimization experiments and ground controls included agarose inside the capillary. The agarose in the base of the GCB is to delay the contact between the precipitant and the protein solutions and to hold the capillaries upright, whereas the agarose in the capillary is necessary to provide a convection-free growth environment. A 1% agarose solution was prepared by adding 100 mg of low-melting-point agarose to 10 mL of crystallization buffer (100 mM TRIS-HCl buffer pH 8.0, 2 mM EDTA, 200 mM NaCl). The suspension was heated in a water bath at 60 °C until homogeneity and the solution was then kept at 37 °C. The GCB was set upright, and the 1% agarose solution poured in the box to a specific height. The gel set in 30 min at room temperature or 2–3 min at -20 °C. After the gel set, a small layer of buffer (100 µL) was poured on top of it. The protein solution, consisting of the specified concentration of TIM and ammonium sulfate in the same buffer, was drawn into the capillaries (diameter 0.5 mm – length 42 mm) by capillarity, keeping the capillary horizontal. For the ground experiments, 0.1% (w/v) agarose gel was also included with the protein solution in the capillary (the 1% agarose solution added to 0.9 volume of protein solution). The capillaries were sealed on top with soft beeswax. Six capillaries were inserted into each GCB and pushed into the gel to the right insertion depth. A layer of precipitant solution (AS) was poured on top of the hardened gel in the GCB base. Finally, the GCB was sealed with vacuum grease and scotch tape. The geometry of the different experiments is summarized in Table 4. Data Collection. The data collection was done at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) on cryocooled crystals. In total, 34 crystals were harvested from the space and ground experiments: 14 (5 space and 9 ground) from the Odissea mission and 20 (11 space and 9 ground) from the Cervantes mission. We tried to select crystals of equivalent size in order to have the same diffracting volume in the subsequent statistical comparison. Just before being flash-cooled, all the crystals were soaked for 1 min in the mother liquor containing 20% glycerol. The diffraction data were collected at 100 K on beamline BM30A (FIP) with the wavelength tuned to 1.078137 Å. All the images were recorded in time mode (∼10 s in the uniform filling mode, 200 mA) on a MAR Research CCD detector placed at a distance of 150 mm from the crystal, corresponding to a maximum resolution of 2.2 Å. The oscillation angle was 0.5° and the slits were set to 0.3 mm. For each data set, a rotation range of 90 ° was collected. All the measurements were indexed and integrated using the program DENZO20 and merged with the program SCALEPACK.20 The protein crystallizes in the space group P3212 with unit-cell parameters: a ) b ) 215 Å, c ) 106 Å, γ ) 120°. One large crystal grown in microgravity during the Cervantes mission was exposed to X-rays in order to reach the highest possible resolution. X-ray data to 1.7 Å resolution were collected at 100 K at the EMBL DESY

2166 Crystal Growth & Design, Vol. 7, No. 11, 2007 synchrotron facility in Hamburg (Germany) using the BW7B beamline equipped with a 30 cm Mar-Research imaging plate scanner. The beamline was operating in dose mode with the wavelength set to 0.83 Å. Prior to exposure, the crystal was flash-cooled in the same conditions as for the previous experiments at ESRF. To avoid spot overlaps at high resolution, a total of 575 images was recorded with an oscillation angle of 0.1° for the 500 first images and 0.6° for the following ones. All the data were processed and merged with the XDS program package.14 Statistical Analysis. The statistical analysis was done using the program SPSS (SPSS Inc., Chicago). For the comparison of two means, the robust Mann–Whitney test was used. It is a nonparametric test that can be used for small samples with outliers. The disadvantage of this test is that it is less powerful than the widely used t test, for normally distributed populations. As such, the Mann–Whitney test will not easily reject the null hypothesis of equal means. All correlations, including the Pearson and Spearman correlations, were calculated with the same program.

Acknowledgment. We are grateful to all the people that have been involved in the Cervantes and Odissea missions. We thank the European Space Agency (ESA) for the flight opportunities. This research was financed by ESA in the context of Prodex projects C90035 and AO2004. The authors gratefully acknowledge the access to the ESRF BM30A beamline. We thank the European Community for Access to Research Infrastructure Action for the Improving Human Potential Programme at the EMBL Hamburg Outstation, Contract HPRI-1999-CT-00017. We also express our thanks to Olivier Minster and Eric Istasse at ESA, to people from LEC (Laboratorio de Estudios Cristalográficos) in Granada, to Frank Dubois and his group from the Free University of Brussels (ULB), and to the B.USOC (Belgian User Support Operation Center) in Brussels. We acknowledge the useful suggestions of the referees and of Klaas Decanniere.

Abbreviations Used TIM ISS GCB AS ESA APCF

triose phosphate isomerase International Space Station Granada Crystallization Box ammonium sulfate European Space Agency Advanced Protein Crystallization Facility

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CG700687T